The present invention relates to novel, rigidified compounds and uses thereof and, more particularly, to uses thereof for modulating the activity of heparanase and hence in the treatment of heparanase-associated diseases and disorders, to uses thereof for modulating the activity of heparin-binding proteins and hence in the treatment of heparin-binding proteins-associated diseases and disorders as well as to uses thereof in the treatment of medical conditions that are treatable by rhodanine or a rhodanine analog.
Proteoglycans (PGs):
Proteoglycans (previously named mucopolysaccharides) are remarkably complex molecules and are found in every tissue of the body. They are associated with each other and also with the other major structural components such as collagen and elastin. Some PGs interact with certain adhesive proteins, such as fibronectin and laminin. The long extended nature of the polysaccharide chains of PGs, the glycosaminoglycans (GAGs), and their ability to gel, allow relatively free diffusion of small molecules, but restrict the passage of large macromolecules. Because of their extended structures and the huge macromolecular aggregates they often form, they occupy a large volume of the extracellular matrix relative to proteins (Murry R K and Keeley F W; Biochemistry, Ch. 57. pp. 667-85).
Heparin Sulfate Proteoglycans (HSPGs):
HSPGs are acidic polysaccharide-protein conjugates associated with cell membranes and extracellular matrices. HSPGs bind avidly to a variety of biologic effector molecules, including extracellular matrix components, growth factor, growth factor binding proteins, cytokines, cell adhesion molecules, proteins of lipid metabolism, degradative enzymes, and protease inhibitors. Owing to these interactions, HSPGs play a dynamic role in biology; in fact most functions of the proteoglycans are attributable to the heparan sulfate (HS) chains, contributing to cell-cell interactions and cell growth and differentiation in a number of systems. HS maintains tissue integrity and endothelial cell function. It selves as an adhesion molecule and presents adhesion-inducing cytokines (especially chemokines), facilitating localization and activation of leukocytes. HS modulates the activation and the action of enzymes secreted by inflammatory cells. The functions of HS changes during the course of the immune response are due to changes in the metabolism of HS and to the differential expression of and competition between HS-binding molecules. (Selvan R S et al; Ann. NY Acad. Sci. 1996, 797: 127-39).
HSPGs are also prominent components of blood vessels (Wight T N et al; Arteriosclerosis, 1989, 9: 1-20). In large vessels HSPGs are concentrated mostly in the intima and inner media, whereas in capillaries HSPGs are found mainly in the subendothelial basement membrane, where they support proliferating and migrating endothelial cells and stabilize the structure of the capillary wall. The ability of HSPGs to interact with extracellular matrix (ECM) macromolecules such as collagen, laminin and fibronectin, and with different attachment sites on plasma membranes suggests a key role for this proteoglycan in the self-assembly and insolubility of ECM components, as well as in cell adhesion and locomotion.
Heparanase—A GAGs Degrading Enzyme:
Degradation of GAGs is carried out by a battery of lysosomal hydrolases. One important enzyme involved in the catabolism of certain GAGs is heparanase. It is an endo-β-glucuronidase that cleaves heparan sulfate at specific interchain sites.
The enzymatic degradation of glycosaminoglycans is reviewed By Ernst et al. (Critical Reviews in Biochemistry and Molecular Biology, 30(5):387-444 (1995). The common feature of GAGs structure is repeated disaccharide units consisting of a uronic acid and hexosamine. Various GAGs differ in the composition of the disaccharide units and in type and level of modifications, such as C5-epimerization and N or O-sulfation. Sulfated GAGs include heparin, heparan sulphate, chondroitin sulphate, dermatan sulphate and keratan sulphate. Heparan sulphate and heparin are composed of repeated units of glucosamine and glucuronic/iduronic acid, which undergo modifications such as C5-epimerization, N-sulfation and O-sulfation. Heparin is characterized by a higher level of modifications than heparan sulphate.
GAGs can be depolymerized enzymatically either by eliminative cleavage with lyases (EC 4.2.2.-) or by hydrolytic cleavage with hydrolases (EC 3.2.1.-). Often, these enzymes are specific for residues in the polysaccharide chain with certain modifications. GAGs degrading lyases are mainly of bacterial origin. In the eliminative cleavage, C5 hydrogen of uronic acid is abstracted, forming an unsaturated C4-5 bond, whereas in the hydrolytic mechanism a proton is donated to the glycosidic oxygen and creating an O5 oxonium ion followed by water addition which neutralizes the oxonium ion and saturates all carbons (Linhardt et al. 1986, Appl. Biochem. Biotech. 12:135-75). The lyases can only cleave linkages on the non-reducing side of the of uronic acids, as the carboxylic group of uronic acid participates in the reaction. The hydrolases, on the other hand, can be specific for either of the two bonds in the repeating disaccharides. In pages 414 and 424 of the review, tables 8 and 14, Ernst et al. list the known GAG degrading enzymes. These tables describe substrate specificity, cleavage mechanism, cleavage linkage, product length and mode of action (endo/exolytic). Heparanase is defined as a GAG hydrolase which cleaves heparin and heparan sulphate at the β1,4 linkage between glucuronic acid and glucosamine. Heparanase is an endolytic enzyme and the average product length is 8-12 saccharides. The other known heparin/heparan sulphate degrading enzymes are beta-glucuronidase, alpha-L iduronidase and alpha-N acetylglucosaminidase, which are exolytic enzymes, each one cleaves a specific linkage within the polysaccharide chain and generate disaccharides. In table 8 the authors list two heparanases; platelet heparanase and tumor heparanase, which share the same substrate and mechanism of action. These two were later on found to be identical at the molecular level (Freeman et al. Biochem J. (1999) 342, 361-268, Vlodavsky et al. Nat. Med. 5(7):793-802, 1999, Hullet et al. Nat. Med. 5(7):803-809, 1999).
Heparin and heparan sulphate fragments generated via heparanase catalyzed hydrolysis are inherently characterized by saturated non-reducing ends, derivatives of N-acetyl-glucosamine. The reducing sugar of heparin or heparan sulphate fragments generated by heparanase hydrolysis contains a hydroxyl group at carbon 4 and it is therefore UV inactive at 232 nm.
Interaction of T and B lymphocytes, platelets, granulocytes, macrophages and mast cells with the subendothelial extracellular matrix (ECM) is associated with degradation of heparan sulphate by heparanase activity. The enzyme is released from intracellular compartments (e.g., lysosomes, specific granules) in response to various activation signals (e.g., thrombin, calcium ionophore, immune complexes, antigens and mitogens), suggesting its regulated involvement in inflammation and cellular immunity. (Vlodavsky I et al; Invasion Metas. 1992; 12(2): 112-27). In contrast, various tumor cells appear to express and secrete heparanase in a constitutive manner in correlation with their metastatic potential. (Nakajima M et al; J. Cell. Biochem. 1988 February; 36(2):157-67). Important processes in the tissue invasion by leukocytes include their adhesion to the luminal surface of the vascular endothelium, their passage through the vascular endothelial cell layer and the subsequent degradation of the underlying basal lamina and extracellular matrix with a battery of secreted and/or cell surface protease and glycosidase activities. Cleavage of HS by heparanase may therefore result in disassembly of the subendothelial ECM and hence may play a decisive role in extravasation of normal and malignant blood-borne cells (Vlodavsky I et al; Inv. Metast. 1992, 12: 112-27, Vlodavsky I et al; Inv. Metast. 1995, 14: 290-302).
It has been previously demonstrated that heparanase may not only function in cell migration and invasion, but may also elicit an indirect neovascular response (Vlodavsky I et al; Trends Biochem. Sci. 1991, 16: 268-71). The ECM HSPGs provide a natural storage depot for β-FGF. Heparanase mediated release of active β-FGF from its storage within ECM may therefore provide a novel mechanism for induction of neovascularization in normal and pathological situations (Vlodavsky I et al; Cell. Molec. Aspects. 1993, Acad. Press. Inc. pp. 327-343, Thunberg L et al; FEBS Lett. 1980, 117: 203-6). Degradation of heparan sulphate by heparanase results in the release of other heparin-binding growth factors, as well as enzymes and plasma proteins that are sequestered by heparan sulphate in basement membranes, extracellular matrices and cell surfaces. (Selvan R S et al; Ann. NY Acad. Sci. 1996, 797: 127-39).
Expression of Heparanase DNA in Animal Cells:
Stably transfected CHO cells express the human heparanase gene products in a constitutive and stable manner. Several CHO cellular clones are particularly productive in expressing heparanase, as determined by protein blot analysis and by activity assays. Although the heparanase DNA encodes for a large 543 amino acids protein (expected molecular weight about 65 kDa, SEQ ID NO: 8) the results clearly demonstrate the existence of three proteins, one of about 60 kDa (H60, SEQ ID NO: 34), another of about 45 kDa (H45, SEQ ID NO: 33) and yet another one of about 8 kDa (H8, SEQ ID NO: 35). It was found that active heparanase is a mature processed form with an apparent molecular weight of 53 kDa (H53), proteolitically cleaved from the latent heparanase precursor of about 60 kDa. This proteolytic cleavage occurs at two cleavage sites Glu109-Ser110 (SEQ ID NO: 1) and Gln157-Lys158 (SEQ ID NO: 2), yielding a 8 kDa polypeptide at the N-terminus, a 45 kDa polypeptide at the C-terminus and a 6 kDa linker polypeptide (H6, SEQ ID NO: 36) that is released due to the cleavage. The formation of the heterodimer between the 8 and 45 kDa subunits is essential for heparanase enzymatic activity (M B Fairbanks et al. J. Biol. Chem. 274, 29587, 1999).
Further details pertaining to heparanase, heparanase gene and their uses can be found in, for example, PCT/US99/09256; PCT/US98/17954; PCT/US99/09255; PCT/US99/25451; PCT/IL00/00358; PCT/US99/15643; PCT/US00/03542; PCT/US99/06189; PCT/US00/03353; PCT/US00/03542; PCT/IL01/00830; PCT/IL01/00950' PCT/IL01/00864; PCT/IL01/01169 and PCT/IL02/00362; and in U.S. Pat. Nos. 6,242,238; 5,968,822; 6,153,187; 6,177,545; and 6,190,875, the contents of all of which are hereby incorporated by reference.
Heparanase Activation:
Heparanase maturation involves the removal of the signal peptide, transforming the 65 kDa pre-pro-heparanase into a 60 kDa pro-heparanase (also referred to herein as latent heparanase or mature heparanase). The 60 kDa latent/mature heparanase is activated into an active heparanase as follows: The 60 kDa latent/mature heparanase is proteolytically cleaved twice into a 45 kDa major subunit, a 8 kDa small subunit and a 6 kDa linker that links the 45 kDa major subunit and the 8 kDa small subunit in the latent enzyme. The 45 kDa major subunit and the 8 kDa small subunit hetero-complex to form the 53 kDa active form of heparanase.
The nature of the protease(s) responsible for activating heparanase is yet unknown.
It will, nevertheless, be appreciated that by modulating the activity of these proteases one can modulate the rate of heparanase activation, hence the rate of heparanase activity and hence the rate of biological processes which depend on heparanase activity.
There is thus a widely recognized need for and it would be highly advantageous to have compounds which can efficiently modulate heparanase activation, by e.g., inhibiting or increasing heparanase activation.
Rhodanine-Based Compounds as Heparanase Inhibitors:
In U.S. patent application Ser. No. 10/916,598, filed Aug. 12, 2004, by the present assignee, which is incorporated by reference as if fully set forth herein, it is taught that heparin plays a critical role in the activation of pro-heparanase and that use of, or interference with, any one of the components or processes involved in heparanase activation may be enough to modulate biological processes, which are governed by heparanase activity. A cell-based assay, which allows the identification of numerous inhibitors of heparanase activation is further taught in this patent application. Based on the above, a comprehensive list of inhibitors, which may be used for inhibiting heparanase activation, was disclosed.
U.S. patent application Ser. No. 10/916,598 particularly teaches several families of compounds that are capable of interfering with heparanase activity. These include, for example, a family of rhodanine analogs, a family of planar aromatic molecules and a family of peptidomimetic molecules.
Rhodanine is a five-membered heterocyclic compound having the following structure:

U.S. patent application Ser. No. 10/916,598 also teaches that inhibition of heparin binding to pro-heparanase may be effected by a heparin-binding agent (or heparan-sulphate binding agent) or by a pro-heparanase binding agent.
Preferred pro-heparanase binding agents, according to the teachings of U.S. patent application Ser. No. 10/916,598, can be collectively represented by the general formula:
wherein:
X is O, S, NR4 or NR5—C(=D);
Y, Z and D are each independently O, S or NR4;
R1 is selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, allyl, aryl, heteroaryl, heteroalicyclic and an acid-containing moiety; and
at least one of R2 and R3 being a substituted or unsubstituted aryl or heteroaryl,
and further wherein:
R4 and R5 are each independently selected from the group consisting of hydrogen, alkyl, cycloalkyl and a aryl.
Particularly promising pro-heparanase binding agents, according to the teachings of U.S. patent application Ser. No. 10/916,598, were found to be rhodanine derivatives having a rhodanine skeleton, such that in the general formula above X is S; Y is O; and Z is S.
It was found, however, that derivatives of rhodanine analogs also act as potent pro-heparanase binding agents. Representative example of rhodanine analogs include, without limitation, compounds having the general formula above, in which X is S; Y is O; and Z is O, and in which X is NR5—C=D; Y is O; Z is O or S; and D is O or S (2-thio/oxo-dihydro-pyrimidine-4,6-dione).
It was further found that another component which may affect the binding potency of these compounds is the substituent R1. Thus, it was found that derivatives of rhodanine or rhodanine analogs in which R1 in the general Formula above is an acid-containing moiety, or a heteroaryl such as, for example, terahydrothiophenyl-1,1-dioxide and 1,5-dimethyl-2-phenyl-1,2-dihydro-3-one-pyrazolyl. Preferred acid-containing moieties were found to include aliphatic carboxylic acid residues having a chain of 2-6 carbon atoms.
More particular promising pro-heparanase binding agents, according to the teachings of U.S. patent application Ser. No. 10/916,598, were found to be derivatives of rhodanine or rhodanine analogs, as shown in the general Formula above, which are substituted by a methylidene group, which in turn, is substituted by a heteroaryl such as furan. The furan is preferably substituted by an aryl group such as a substituted phenyl. Suitable substituents of the phenyl ring include, for example, hydrogen, alkyl, hydroxy, thiohydroxy, alkoxy, thioalkoxy, halo, nitro, trihaloalkyl, C-carboxy, O-carboxy, C-amido, N-amido, S-sulfonamido and N-sulfonamido, or, alternatively, at least two substituents form a five- or six-membered cyclic, heteroalicyclic, aromatic or heteroaromatic ring. Preferred substituents are hydrogen, halo (e.g., chloro) and/or nitro.
Furthermore, the nature of the substituents on the phenyl ring was found to affect the binding potency of these agents. Hence, the substituents at the ortho positions with respect to the furan are preferably hydrogen, and/or an electron donating-group such as alkyl, cycloalkyl, hydroxy, alkoxy, thiohydroxy, thioalkoxy, aryloxy and thioaryloxy, whereby the substituent at the meta and para positions are preferably hydrogen and/or an electron-withdrawing group such as halo, nitro, trihaloalkyl and C-carboxy. The C-carboxy substituent is preferably a carboxylic acid group.
The most promising agents, according to the teachings of U.S. patent application Ser. No. 10/916,598, can therefore be collectively represented by the following general Formula:

wherein X, Y, Z and R1 are as described above, W is O or S, defining a furan ring or a thiophene ring; R2, R7 and R8 are each independently hydrogen, alkyl, cycloalkyl, aryl and heteroaryl, preferably hydrogen; and R10 to R14 are each independently a substituent as described hereinabove.
Some of the compounds described above as pro-heparanase binding agents were found to have a dual activity, such that in addition to inhibiting pro-heparanase activation, they inhibit heparanase activity. Preferred compounds in this category are those bearing a carboxylic acid group, either as the R1 substituent or as one of the R10-R14 substituents.
The most preferred compounds according to the teachings of U.S. patent application Ser. No. 10/916,598, can be described by the general formula:
wherein:
R1 is selected from the group consisting of hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted allyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heteroalicyclic and an acid-containing moiety having the general formula:—(CH2)n-CH(R6)-Q1(OH),
whereas,
n is integer that equals 0-20;
R6 is selected from the group consisting of hydrogen, alkyl and Q2(OH); and
Q1 and Q2 are each independently selected from the group consisting of C═O and S(═O)2; and
R10-R14 are each independently selected from the group consisting of halo, nitro, alkoxy, aryloxy, hydroxy, thiohydroxy, thioalkoxy, thioaryloxy, alkyl, aryl, heteroaryl, heteroalicyclic, trihaloalkyl, C-carboxy, O-carboxy, oxo, C-amido, N-amido, S-sulfonamido and N-sulfonamido,
whereby either R1 is the acid-containing moiety or at least one of the R10-R14 is C-carboxy.
Alternatively, both R1 and one or more of the substituents R10-R14 include an acidic moiety.
Although the above-described compounds were found effective in modulating heparanase activity and heparin-binding proteins activity, the present inventors have envisioned that structurally-related compounds which are characterized by a lower number of free-to-rotate bonds and hence by a rigidified structure could exhibit higher affinity to the target and thus would exhibit improved performance.
Rhodanine-based compounds are also known as efficient agents for treating a wide scope of other medical conditions. These include, for example, CNS disorders such as Alzheimer's disease and schizophrenia, atherosclerosis, autoimmune diseases, bacterial infections such as anthrax, cholera, and tuberculosis, blood coagulation, bone disorders, cancer, cardiovascular diseases, diabetes, fungal infections, gastro-intestinal disorders, hair loss, hypercholesterolemia, inflammation, pain, and viral diseases and infections such as hepatitis C, herpes, HIV, and smallpox.
The wide scope of medical conditions that is treatable by rhodanine-based compounds is indicative of the beneficial merits of such compounds as a concept for drug development in general. However, such a wide scope of activities may also imply that rhodanine-based compounds might exert toxic and other adverse effects due to lack of specificity.
The present inventors have therefore further envisioned that rhodanine-based compounds which are characterized by a lower number of free-to-rotate bonds and hence by a rigidified structure would exhibit higher specificity to the targeted organ or system and could further be efficiently utilized in the treatment of these conditions.
While some rhodamine-based compounds that have a rigidified structure have been reported, the biological activity of these compounds has been questionable. Thus, compounds having a N-(3-morpholino)propyl rhodanine analog residue being covalently attached to a 5-(3-methoxyphenyl)thiophene group and a 5-(3-nitrophenyl)thiophene group have been disclosed by Carter et al. (in Proc. Natl. Acad. Sci., 98, 11879, 2001). These compounds, along with other, non-rigid rhodanine-based compounds, were tested for their binding to tumor necrosis factor-alpha (TNF-alpha), and were found active only when exposed to light. 5-Arylidene-2-thioxodihydropyrimidine-4,6(1H,5H)-diones and 3-thioxo-2,3-dihydro-1H-imidazolo[1,5-a]indol-1-ones were also reported to act as light-dependent TNF-alpha antagonists (Voss et al., Bioorg. Med. Chem. Lett., 13, 533-538, 2003).
The light-dependency activity of these compounds as TNF-alpha antagonists suggests that the compounds disclosed in these publications are artifacts, having artificial properties that cannot be exhibited in a human body (where there is no light). Thus, no definite biological activity of rigid rhodanine-based compounds in taught in these publication. In addition, while these publications teach the artificial binding of these compounds to TNF-alpha, these publications are completely silent with respect to the activity of such compounds as modulators of heparanase activity, of heparin-binding proteins activity and as active agents that affect other biological pathways.
There is thus a widely recognized need for, and it would be highly advantageous to have, novel rhodanine-based compounds, having a rigidified structure, which could be efficiently utilized for modulating heparanase and/or heparin-binding proteins activities, as well as other biological processes, preferably in a non light-dependent manner.